Translocation of the SBP-tagged N-domain is arrested by SAv
To arrest N-domain translocation, a 38-residue SBP-tag was fused to the N terminus of mouse synaptotagmin II (SytII) (, S-I). The model protein construct consisted of a 38-residue N-terminal SBP-tag, a 70-residue hydrophilic sequence (7-residue glycosylation probe sequence, 4 residues encoded by restriction enzyme sites, and a 59-residue SytII N-terminal domain; for details see Materials and methods), the 27-residue H-segment of the SA-I sequence, and the cytoplasmic domain of SytII (). The two glycosylation sites in the N-terminal hydrophilic domain were used as an indicator of translocation because glycosylation occurs only in the ER lumen. To produce defined translocation intermediates, we used truncated mRNAs for cell-free synthesis. Because they did not possess an in-frame termination codon, the synthesized nascent chain remained on the ribosome as a peptidyl tRNA to form the ribosome-nascent chain complex. When mRNA, truncated at Arg200 of SytII, was translated for 60 min, the S-I protein product was efficiently diglycosylated in the presence of rough microsomal membrane (RM) (, lane 2). There was little of the monoglycosylated form. Endoglycosidase H (EndoH) treatment caused a downward shift of the top band, confirming that the top band is glycosylated (lane 3). When translated in the presence of SAv, glycosylation was suppressed (lane 5). In contrast, SAv did not affect glycosylation in the presence of biotin (lane 4), indicating that N-domain translocation was arrested by a specific interaction of SBP-tag with SAv. To examine whether the arrested translocation can be resumed, biotin was added after translation was performed in the presence of SAv and terminated with cycloheximide (CHX). Upon addition of biotin, glycosylation efficiently resumed (lane 6). In the absence of biotin, little glycosylation occurred, even after a 60-min chase (lane 7). These results indicate that N-domain translocation via the SA-I sequence was arrested by SAv and could be chased by biotin.
Figure 1. Two intermediates of N-domain translocation generated by SBP-tag trapping. (A) The SBP-tag (SBP) and the glycosylation sequence were fused to the N terminus of SytII (S-I). A 38-residue spacer sequence was inserted between the glycosylation probe sequence (more ...)
To insert the polypeptide chain further into the translocon, a 38-residue sequence was inserted as a spacer between the SBP-tag and SA-I sequence (, S-38-I). This 38-residue sequence was derived from the lumenal loop of human anion exchanger 1, which is often used as a passenger sequence for translocation experiments (Sato et al., 2002
; Kida et al., 2005
). When translated in the presence of RM and SAv, the monoglycosylated form was predominantly observed (, lane 3). When the reaction was chased in the presence of biotin, both the nonglycosylated and monoglycosylated forms were converted to the diglycosylated form (lane 4). When the former glycosylation site was silenced, the monoglycosylated intermediate form was not affected (unpublished data), indicating that the first glycosylation site of the intermediate was still on the cytoplasmic side and the latter glycosylation site was in the lumen (). The 38-residue spacer resulted in the hydrophilic sequence forming a TM disposition (). Collectively, these findings indicate that we created two different intermediates of N-domain translocation: in one intermediate, the SA-I sequence and hydrophilic segment spanned the membrane (termed “advanced stage”); whereas in the other intermediate, the SA-I sequence was at an earlier stage of translocation (termed “earlier stage”) ().
N-domain translocation resumes even in the presence of a second translocating segment
We then examined whether insertion of a downstream polypeptide chain is influenced by the N-domain translocation intermediates. The TM3 segment of human Na+
exchanger isoform 6, which mediates membrane insertion of its following portion (Miyazaki et al., 2001
), was positioned as the second insertion sequence (, II). A third glycosylation site and a prolactin sequence were fused as a reporter. When the S-I-II protein was synthesized in the presence of RM, the triglycosylated form was mainly observed (, lane 2). EndoH treatment confirmed glycosylation of the top bands (lane 3). When synthesized in the presence of SAv, triglycosylation was suppressed and the monoglycosylated form was observed as the main product (lane 4). When chased in the presence of biotin, the triglycosylated form became the major product (lane 5). In the absence of biotin, 60-min incubation induced little glycosylation (lane 6).
Figure 2. Earlier intermediate does not influence the next insertion. (A) The SA-I sequence in the S-I protein was followed by the second H-segment (II) of the TM3 in human Na+/H+ exchanger isoform 6, the glycosylation probe sequence (third open (more ...)
To confirm the identity of the glycosylated sites, the second glycosylation site was silenced by a single point mutation (, S-I-II(2G)). The diglycosylated form was observed as the major product instead of the triglycosylated form (lanes 8 and 11), whereas the monoglycosylated form observed in the presence of SAv was not affected (lane 10). Only the third glycosylation site was thus glycosylated in the presence of SAv ().
Collectively, when N-domain translocation was arrested by SAv, the third glycosylation site after the second H-segment was translocated into the lumenal side of the RM. In this situation, the SA-I sequence should occupy the signal recognition site of the Sec61 complex, while the second H-segment could be inserted and mediate the following translocation. It should be noted that the N-domain polypeptide chain could move across the membrane, even when the second translocating hydrophilic segment spanned the membrane.
Two translocating segments can span the membrane
To form the advanced intermediate stage of the S-I-II protein, the 38-residue spacer sequence was inserted between the SBP-tag and SA-I sequences (, S-38-I-II). When synthesized in the absence of SAv, the triglycosylated form was observed depending on the RM (, lane 2). When translated in the presence of SAv, the diglycosylated form was mainly observed (lane 3), indicating that the second and third positions of the main product were in the lumen and only the N-terminal position was on the cytoplasmic side (). The glycosylation of the N-terminal site was chased by the post-translational addition of biotin (, lane 4). This finding demonstrates that the N-domain of the S-38-I-II protein was in the advanced intermediate stage (). As shown in the next section, the C-terminus of this intermediate was in the ribosome. The two hydrophilic segments were simultaneously in the TM disposition (). The former translocating segment did not interfere with the insertion of the second polypeptide chain and the second segment did not affect the resumption of N-domain translocation.
Figure 3. Two translocating hydrophilic segments span the membrane. (A) The SBP-tag of S-I-II was separated from SA-I by a 38-residue spacer (S-38-I-II). As a third H-segment, 15 residues in the indicated region were replaced with 15 leucine residues (S-I-II-15L). (more ...)
To examine the effect of an H-segment in the C-terminal chain on the translocation intermediate stage, 15 amino acid residues in the second translocating segment were exchanged with 15 leucine residues (, 15L constructs). The 15-leucine sequence was followed by 50 residues, so that the segment should exit the ribosome and enter the translocon. Even in the presence of the 15-leucine segment, essentially the same results were obtained as with S-I-II and S-38-I-II (, lanes 5–12). When synthesized in the absence of SAv, the triglycosylated form was mainly observed (lanes 6 and 10). In the presence of SAv, N-domain translocation was suppressed and the monoglycosylated form of S-I-II-15L (lane 7) and the diglycosylated form of S-38-I-II-15L (lane 11) were the main products. Triglycosylation was observed after a 60-min chase in the presence of biotin (lanes 8 and 12), indicating that N-domain translocation was chased in both cases. The third H-segment (15L) did not the affect resumption of N-domain translocation ().
Productivity of translocation intermediates
To ascertain the fate of the C-terminal segment, a fourth glycosylation site was created 40 residues from the truncation site (). This glycosylation site should be accessible only when the C-terminal segment is released from the ribosome and translocated through the membrane. In the presence of CHX, the diglycosylated form was converted to the triglycosylated form by biotin (, lane 2). In contrast, when treated with puromycin (Puro), there was a significant increase in the triglycosylated form before the biotin chase (, compare lanes 1 and 3). After a 60-min biotin chase, the intermediate forms were converted to the tetraglycosylated form (lane 4). The fourth glycosylation site became accessible to the glycosylation enzyme in the presence of Puro (). When the 15-leucine segment was present, tetraglycosylation was suppressed (, compare lanes 4 and 8), indicating that the 15-leucine segment stopped translocation of the C-terminal chain. The C terminus of the S-38-I-II protein was trapped in the ribosome and C-terminal translocation can be chased as efficiently as N-terminal translocation. The resumption of N-domain translocation was also not affected by C-terminal release from the ribosome.
Figure 4. Productivity of the translocation intermediates. (A) The fourth glycosylation site was generated 40 residues away from the C-terminal truncation site of (4G) constructs. (B) After translation was performed for 60 min, the reaction was terminated by CHX (more ...)
In contrast to the results with the advanced intermediate of the S-38-I-II protein, the resumption of N-domain translocation in an earlier stage of the S-I-II protein was significantly affected by Puro treatment (, compare lanes 2 and 4). Even in the earlier intermediate stage, the SA-I and N-terminal domain remain actively engaged with the translocon, depending on the ribosome, during the translocation arrest. The differential influence of Puro indicates that SA-I sequences in the earlier and advanced stages are in different states.
Using the S-38-I-II(4G) model protein, we confirmed membrane topology of the two TM segments. The loop between two TM segments is on the cytoplasmic side and accessible to the externally added proteinase K (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200707050/DC1
Each translocating hydrophilic segment flanks Sec61α
To probe proteins adjacent to the translocating polypeptides, we performed chemical cross-linking experiments. Two cysteine residues were created in either the first or second translocating hydrophilic segment using a Cys-less mutant (see Materials and methods) (). The Cys mutants were synthesized in the presence of RM and SAv, and the cross-linking reaction was performed with a homobifunctional cross-linker BMH, whose spacer is 16.1 Å. Cross-linked products were subjected to immunoprecipitation with anti-Sec61α antibody (). A Cys-residue at positions-1, -2, -3, -4, -7, or -8 gave a significant cross-linked band of 90 kD that was immunoreactive with anti-Sec61α antibody. Given that the probe is ~50 kD (, asterisk), the size of the cross-linking partner is consistent with that of Sec61α. The negligible and weak cross-linking of Cys-residues at positions-5 and -6 indicate the position specificity of the cross-linking reaction. The immunoreactive cross-linked products were not observed when incubated in the absence of a cross-linker (, lanes 26 and 28). Essentially the same results were obtained using other cross-linkers with shorter spacers, bismaleimidoethane (BMOE; 8.0 Å), 1,4-bismaleimidobutane (BMB; 10.9 Å), and N,N-(methylene-4-1-phenylene)bismaleimide [BM(PEO)2
; 14.7 Å] (Fig. S2). The immunoreactive band of the position-4 Cys mutant was not observed with unrelated antibodies, anti-Sec63 and anti-SAv (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200707050/DC1
). These results indicate that both translocating hydrophilic chains flanked the Sec61 channel. It is likely that the nascent chain existed within Sec61 pore. The polypeptide might be outside the small Sec61 pore and still adjacent to Sec61 subunit. We performed the same cross-linking experiment using the construct in which Cys-residues were included in both translocating hydrophilic segments and found a faint but significant super-shifted band in addition to the band cross-linked with one Sec61α (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200707050/DC1
), suggesting that two Sec61α can be cross-linked with the single nascent polypeptide chain. The exact nature of the super-shifted molecule, however, remains to be examined.
Figure 5. Two translocating polypeptides flank Sec61α. (A and B) For site-specific chemical cross-linking, two Cys residues were created by point mutations at the indicated positions (numbered) using a Cys-less mutant of S-38-I-II. (C) The Cys mutants were (more ...)
We then examined the effect of biotin or Puro treatments on the environment of the integration intermediates. Cross-linking in the position-3 Cys mutant was little affected by Puro, but was abolished by biotin (, lanes 9 and 12). Cross-linking with the position-7 Cys residues in the presence of Puro was more diminished than that with position-3 (, lanes 9 and 21). However, the residual cross-linking of position-7 in the presence of Puro was further decreased after biotin treatment (lane 24). On the other hand, position-7 cross-linking was not affected by biotin chase in the presence of CHX (unpublished data). These results indicate that cross-linking with Sec61α reflects the productive and specific intermediate and that the upstream translocating segment in the advanced intermediate stage continued to flank the Sec61 channel even after Puro treatment. The residual cross-linking of position-7 after Puro treatment in the absence of biotin might suggest that upstream translocating chain affects the downstream translocation.
Multiple TM insertions do not affect N-domain translocation
To examine the effect of the insertion of multiple H-segments on N-domain translocation, the TM segments (TM2–TM7) of bovine rhodopsin were attached downstream of the SA-I sequence (). When synthesized in the presence of RM, the N-terminal site was glycosylated (, lane 2). The glycosylation was arrested in the presence of SAv, but resumed after the biotin chase (lanes 3 and 4). To confirm multiple insertions of the following TM segments, a glycosylation loop sequence was inserted into the lumenal loop between either TM2 and TM3 or TM4 and TM5 (). In both cases, the products were diglycosylated in the presence of RM (, lanes 6 and 10). When translated in the presence of SAv, the monoglycosylated forms were the major products (lanes 7 and 11). These were converted to the diglycosylated forms after the biotin chase (lanes 8 and 12). For unknown reasons, the diglycosylated form of these constructs was occasionally smeared or split (e.g., lanes 6, 8, and 12).
Figure 6. Insertion of six TM segments did not compete with N-domain translocation. (A) Six TM segments of rhodopsin were fused after the SA-I (S-I-Rhod). The endogenous glycosylation site in SytII was silenced. In the G-loop constructions, the glycosylation sequence (more ...)
When the intermediate was treated with Puro instead of CHX, glycosylation after the chase reaction was significantly affected (), indicating that the ribosome is actively involved in maintaining the productive state of the earlier intermediate of N-domain translocation, even after the insertion of six successive TM segments. The insertion of multiple H-segments did not affect the resumption of arrested N-domain translocation. An insertion intermediate of the N-terminal SA-I sequence did not affect the insertion of the following multiple TM segments.